Back to EveryPatent.com
United States Patent |
5,248,594
|
Aloisio
,   et al.
|
September 28, 1993
|
Immunodiagnostic reagent specific for legionella
Abstract
A set of three unique monoclonal antibodies have been produced which
recognize Legionella with particular specificity, without substantial
cross-reactivity with non-Legionella bacteria. These monoclonal antibodies
are useful as immunodiagnostic reagents for detecting Legionella.
Inventors:
|
Aloisio; Carol H. (Norcross, GA);
Carlone; George M. (Stone Mountain, GA);
Plikaytis; Bonnie B. (Tucker, GA);
Sampson; Jackie (College Park, GA)
|
Assignee:
|
The United States of America as represented by the Department of Health (Washington, DC)
|
Appl. No.:
|
548011 |
Filed:
|
July 5, 1990 |
Current U.S. Class: |
435/7.32; 435/960; 435/975; 436/518; 436/804; 530/388.4 |
Intern'l Class: |
G01N 033/569; C07K 015/28 |
Field of Search: |
435/7.32,172.2,240.27
436/548
530/387,388.4
|
References Cited
U.S. Patent Documents
4931547 | Jun., 1990 | Hoffman et al. | 530/387.
|
Foreign Patent Documents |
364971 | Apr., 1990 | EP | 435/7.
|
Other References
K. K. Sethi, "Monoclonal Antibodies Against Legionella pneumophila
Serogroup 1 Antigens: Characterization and Their Potential Applications"
in Monoclonal Antibodies Against Bacteria, pp. 121-136 (1985).
Helsel et al, Current Microbiology vol. 16: 201-208 (1988).
Plikaytis et al, "Purified 60-Kilodalton Legionella Protein Antigen with
Legionella-Specific and Nonspecific Epitopes", J. Clin. Microbiol. 25(11)
pp. 2080-2084 (Nov. 1987).
Nowinski et al, "Monoclonal Antibodies for Diagnosis of Infectious Diseases
in Humans", Science 219 pp. 637-644 (11 Feb. 1983).
Edelstein, "Legionella" in Manual of Clinical Microbiology, 4th Ed. pp.
373-381 (1985).
|
Primary Examiner: Kepplinger; Esther L.
Assistant Examiner: Bidwell; Carol E.
Attorney, Agent or Firm: Lowe, Price, LeBlanc & Becker
Claims
What is claimed is:
1. A monoclonal antibody selected from the group consisting of the
monoclonal antibodies GB5BE8 produced by hybridoma ATCC HB10459, GB5AF6
produced by hybridoma ATCC HB10453, CA4AF5 produced by hybridoma ATC
HB10439, and mixtures thereof, said monoclonal antibody having binding
specificity for detection of Legionella species.
2. The monoclonal antibody of claim 1, which is GB5BE8 produced by
hybridoma ATCC HB10459.
3. The monoclonal antibody of claim 1, which is GB5AF6 produced by
hybridoma ATCC HB10453.
4. The monoclonal antibody of claim 1, which is CA4AF5 produced by
hybridoma ATCC HB10439.
5. A polyvalent mixture of monoclonal antibodies comprising GB5BE8 produced
by hybridoma ATCC HB10459, GB5AF6 produced by hybridoma ATCC HB10453,
CA4AF5 produced by hybridoma ATCC HB10439, said mixture in combination
reacting with the strains of Legionella as set forth in Table 1.
6. An immunodiagnostic reagent for detecting Legionella, comprising an
immunoreactive amount of a monoclonal antibody of claim 1 in a vehicle.
7. An immunodiagnostic reagent according to claim 6, wherein the vehicle is
a sterile, non-toxic medium.
8. A method for screening sample to detect a Legionella species, comprising
contacting a sample suspected of being infected with Legionella with a
monoclonal antibody selected from the group consisting of GB5BE8 produced
by hybridoma ATCC HB10459, GB5AF6 produced by hybridoma ATCC HB10453,
CA4AF5 produced by hybridoma ATCC HB10439, and mixtures thereof, and
detecting specific binding of said monoclonal antibody to the sample,
wherein a positive specific binding reaction is indicative of the presence
of Legionella in said sample, said method not being positively effective
to detect Legionella bozemanii strain Toronto 3, Legionella hackeliae
strain Lansing 2 or strain 798-PA-H.
9. A method according to claim 8, wherein screening is carried out by an
immunohistochemical test, an immunoblotting test, or a radioimmunoassay
test.
Description
The present invention is related generally to immunodiagnostic reagents.
More particularly, the present invention is related to novel monoclonal
antibodies for genus wide detection specifically of Legionella species.
BACKGROUND OF THE INVENTION
Currently, there are about 30 recognized species containing 47 serogroups
of Legionella. Present serological detection methods are based on about 48
serogroup specific antigens which require about 48 serogroup specific
antisera. Sampson et al. (J. Clin. Micro., 23:92-99, 1986) described a
58-kilodalton (later 60-kilodalton) protein antigen that was present in
all Legionella species examined and which was found to be immunogenic in
humans, reacting with serum from 100% of the culture-confirmed cases of
legionellosis that were tested. Antibody to this protein was considered as
a possible probe in immunological procedures to detect all species in the
genus Legionella. Polyclonal rabbit antisera to the 60-kilodalton protein
was produced by the method of Plikaytis et al. (J. Clin. Micro.,
25:2080-2084, 1987) for use as a detection reagent. To achieve specificity
to the Legionella genus, the polyclonal antisera had to be sequentially
absorbed with three heterologous organisms, thereby significantly
decreasing the potency of the antisera. In contrast, the monoclonal
antibodies produced by the hybridoma cell lines are not subject to the
variabilities encountered in the production of polyclonal antisera.
A variety of diagnostic techniques for detection of Legionnaire's disease
are known. Patent No. 87/05609 issued to Hoffman describes monoclonal
antibodies for the detection of Legionella species. However, these
antibodies have cross-reactivity with the bacteria of other genus.
SUMMARY OF THE INVENTION
It is, therefore, an object of the present invention to provide a set of at
least three monoclonal antibodies which, in combination, recognize all
known species of Legionella without substantial cross-reactivity with
other bacteria with which heretofore known monoclonal antibodies exhibit
significant cross-reactivity.
It is another object of the present invention to provide novel
immunodiagnostic reagents for differentially detecting the presence of
bacteria belonging to the genus of Legionella in biological or
environmental samples and the like.
Other objects and advantages will become evident from the following
detailed description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other objects, features and many of the attendant advantages of
the invention will be better understood upon a reading of the following
detailed description when considered in connection with the accompanying
drawings wherein:
FIGS. 1A-1D show binding specificity of Hoffman's monoclonal antibody and
the claimed monoclonal antibodies with the 60 kDa protein among
non-Legionella bacteria using Western blot analysis.
Blots were probed with Hoffman's monoclonal antibody GW2X4B8B2H6 (A),
Aloisio's monoclonal GB5BE8 (B), GB5AF6 (C), and CA4AF5 (D). Lane 1
contains a positive control strain of L. pneumophila serogroup 1; 2,
Acinetobacter lwoffii; 3, Pseudomonas aeruginosa; 4, Bordetella
bronchiseptica; and 5 B. pertussis. Note the strong reactivity in lanes 2,
4, and 5 with Acinetobacter, B. bronchiseptica, and B. pertussis when
probed with the Hoffman antibody.
DETAILED DESCRIPTION OF THE INVENTION
The above and various other objects and advantages of the present invention
are achieved by producing a set of three stable hybridoma clones such that
each clone secretes sufficient amount of uniformly specific monoclonal
antibodies which bind to specific antigenic sites (isotypic epitopes)
unique to the 60-kilodalton protein of Legionella. These three monoclonal
antibodies obtained in accordance with the present invention are
designated herein as GB5BE8, GB5AF6 and CA4AF5.
A deposit of the three hybridomas secreting monoclonal antibodies GB5BE8,
CA4AF5 and GB5AF6 has been made at the ATCC, Rockville, Md., on May 23,
1990, Apr. 24, 1990 and May 10, 1990, respectively under accession numbers
HB 10459, HB10439 and HB10453, respectively. The deposit shall be viably
maintained, replacing if it becomes non-viable during the life of the
patent, for a period of 30 years from the date of the deposit, or for 5
years from the last date of request for a sample of the deposit, whichever
is longer and, upon issuance of the patent, made available to the public
without restriction in accordance with the provisions of the law. The
Commissioner of Patents and Trademarks, upon request, shall have access to
the deposit.
Unless defined otherwise, all technical and scientific terms used herein
have the same meaning as commonly understood by one of ordinary skill in
the art to which this invention belongs. Although any methods and
materials similar or equivalent to those described herein can be used in
the practice or testing of the present invention, the preferred methods
and materials are now described. All publications mentioned hereunder are
incorporated herein by reference. Unless mentioned otherwise, the
techniques employed or contemplated herein are standard methodologies well
known to one of ordinary skill in the art. The materials, methods and
examples are illustrative only and not limiting.
The term "substantial" cross reactivity as defined herein means significant
or considerable amount of cross reactivity.
MATERIALS AND METHODS
Purified 60-kilodalton protein antigen was produced from Legionella
pneumophilia serogroup-1. A highly enriched extract was produced by
ultracentrifugation. Further purification was carried out by standard
affinity chromatography and ion exchange high performance liquid
chromatography. For initial immunization, a dosage of 20 micrograms of
purified protein in phosphate buffered saline with incomplete Freund's
adjuvant was administered to mice. This was repeated at 6 weeks. One week
later, the animal was given 10 micrograms purified protein intravenously
on two consecutive days. The general procedures of Kohler and Milstein
(Nature, 256, 1975) were used as modified by Zola and Brooks (Monoclonal
Antibodies: Techniques and Applications, J. G. R. Hurrell (ed.), CRC
Press, Boca Raton, pp. 1-57, 1982). Immunized mice and antibody from
hybridized cells were screened for reactivity by standard Western blot
analysis using purified 60-kilodalton protein as the antigen and goat
anti-mouse horseradish peroxidase conjugate. From numerous clones tested,
three clones were isolated that exhibited the desired reactivity. Further
analysis of the three clones was done using standard SDS-PAGE followed by
immunoblots on 42 Legionella serogroups (23 species) and 62 non-Legionella
bacterial strains; the results are shown in Table 1. The results indicate
that the three hybridoma cell lines produce monoclonal antibody that, in
combination, react with all tested strains of Legionella.
It should be noted that there was no reactivity with the five strains of
Bordetella and one strain of Acinetobacter tested by Western blot
analysis. This is a significant difference from the reactivity of the
GW2X4B8B2H6 monoclonal antibody reported by Hoffman. Hoffman reported that
this antibody reacts strongly with 12 strains of Bordetella and one strain
of Acinetobacter when tested in an ELISA method. In comparative tests
presented herein, it was found that the GW2X4B8B2H6 antibody strongly
reacted with the Bordetella and Acinetobacter species in Western blot
analysis (FIG. 1). This is quite significant since a monoclonal antibody
or pool of monoclonal antibodies that detect most Legionella species
without substantial cross-reactivity with non-Legionella species would
provide an important screening tool for both clinical and environmental
samples. Clearly, the strong cross-reactivity of the Hoffman monoclonal
antibody makes it unfit for this type of application due to unacceptable
cross-reactivity. The strong cross-reactivity of the GW2X4B8B2H6 Hoffman
antibody and the lack of this cross-reactivity in the monoclonal
antibodies reported herein indicate that even though these antibodies
react to the same 60-kDa protein, the epitope specificity is distinctly
different.
The unique monoclonal antibodies of the present invention now provide a
reliable, rapid, simple and specific diagnostic test for Legionella. The
method comprises reacting a sample suspected of Legionella infection with
a part (reactive fragment) or whole of the monoclonal antibodies of the
present invention, a positive immunological reaction being indicative of
the presence of Legionella bacterium in the sample. Such immunodiagnostic
tests, which include immunohistochemical, immunoblotting, radioimmunoassay
and the like, are quite routine and standard and well known to one of
ordinary skill in the art. A diagnostic kit or an immunodiagnostic reagent
in accordance with the present invention comprises containers containing
an immunoreactive amount of the monoclonal antibodies of the present
invention, either alone or as a combined mixture in a sterile, non-toxic
medium such as a buffered solution and the like.
It is understood that the examples and embodiments described herein are for
illustrative purposes only and that various modifications or changes in
light thereof will be suggested to persons skilled in the art and are to
be included within the spirit and purview of this application and scope of
the appended claims.
TABLE 1
__________________________________________________________________________
SPECIFICITY OF LEGIONELLA 60K MONOCLONALS
GB5 GB5 CA4
Bacteria Serogroup
Strain ID
BE8 AF6 AF5
__________________________________________________________________________
Legionella pneumophila
1 Philadelphia 1
+ + +
Legionella pneumophila
2 Togus 1 + + +
Legionella pneumophila
3 Bloomington 2
+ + +
Legionella pneumophila
4 Portland 1
+ + +
Legionella pneumophila
5 Cambridge 2
+ + +
Legionella pneumophila
6 Chicago 2
+ + +
Legionella pneumophila
7 Chicago 8
+ + +
Legionella pneumophila
8 Concord 3
+ + +
Legionella pneumophila
9 In-23-G2-C2
+ + +
Legionella pneumophila
10 Leiden 1 + + +
Legionella pneumophila
11 797-PA-H + + +
Legionella pneumophila
12 570-CO-H - + +
Legionella pneumophila
13 82A3105 +/- + +
Legionella pneumophila
14 1169-MN-H
+ /-
+ +
Legionella bozemanii
1 WIGA + + +
Legionella bozemanii
2 Toronto 3
- +/- +/-
Legionella dumoffii
1 NY-23 +/- + -
Legionella gormanii
1 LS-13 + + +/-
Legionella micdadei
1 Tatlock +/- + +
Legionella longbeachae
1 Long Beach 4
+ + +/-
Legionella longbeachae
2 Tucker 1 + + +/-
Legionella jordanis
1 BL-540 + + +/-
Legionella oakridgensis
1 Oak Ridge 10
- - +
Legionella wadsworthii
1 81-716 + + -
Legionella feeleii
1 WO-44C-C3
- - +
Legionella feeleii
2 691-WI-H - - +
Legionella sainthelensi
1 Mt.St.Helen 4
+ + +
Legionella anisa
1 WA-316-C3
+ + +/-
Legionella santicrucis
1 SC-63-C7 - + +
Legionella steigerwaltii
1 SC-18-C9 + + +/-
Legionella parisiensis
1 PF-209C-C2
+ + +
Legionella spiritensis
1 Mt.St.Helen 9
+ + +
Legionella hackeliae
1 Lansing 2
- - +/-
Legionella hackeliae
2 798-PA-H - - +/-
Legionella maceachernii
1 PX-1-G2-E2
+/- +/- +
Legionella jamestowniensis
1 JA-26-G1-E2
+/- + +
Legionella cherrii
1 ORW + + +/-
Legionella rubrilucens
1 WA-270A-C2
+ + +
Legionella erythra
1 SE-32A-C8
+/- +/- -
Legionella israelensis
1 Bercovier 4
- - +
Legionella birminghamensis
1 1407-AL-H
+/- +/- +
Legionella cincinnatiensis
1 72-OH-H + + +/-
Pseudomonas testosteroni
KC1765 - - -
Yersinia pseudotuberculosis
514-84 - - -
Shigella sonnei 0 85 - - -
Pseudomonas aeruginosa
5 - - -
Streptococcus pneumoniae
Pn-1 - - -
Pseudomonas maltophilia
KC1768 - - -
Bordetella bronchisepta
F6286 - - -
Bordetella bronchisepta
F6287 - - -
Pseudomonas diminuta KC1797 - - +/-
Pseudomonas cepacia KC1766 - - -
Serratia marcescens 4391-83 - - -
Escherichia coli 16 - - -
Serratia marcescens 4391-83 - - -
Escherichia coli 16 - - -
Hemophilus influenzae a
KC818 - - -
Pseudomonas fluorescens
CDC93 - - -
Escherichia coli 013 - - -
Klebsiella ascorbata 426-84 - - -
Escherichia fergusonii
1295-83 - - -
Shigella flexneri 37 - - -
Staphylococcus aureus 42BP - - -
Yersinia enterocolitica
1149-84 +/- +/- -
Hemophilus influenzae e
KC528 - - +/-
Hemophilus influenzae b
1179-85 - - +/-
Pseudomonas aeruginosa
2 - - -
Pseudomonas aeruginosa
0 8 - - -
Klebsiella oxytoca 4698-84 - - -
Escherichia hermanii 460-84 - - -
Providencia stuartii 4007-83 - - -
Providencia rettgeri 5317-81 - - -
Enterobacter aerogenes
1942-81 - - -
Alcaligenes faecalis - - -
Acinetobacter lwoffii mima - - -
Klebsiella pneumoniae 4809-84 - - -
Pseudomonas paucimobilis
B3271 - - -
Bordetella pertussis F6324 - - -
Bordetella pertussis F6323 - - -
Pseudomonas alcaligenes
ABB50 - - -
Pseudomonas acidovorans
KC1769 - - -
Neisseria meningitidis
C KC792 - - -
Pseudomonas fluorescens
E.B. - - -
Bordetella parapertussis
E1142 - - -
Flavobacterium meningosepticum
0 698 - - -
Listeria monocytogenes
KC1775 - - -
Listeria monocytogenes
KC2380 - - -
Listeria innocua KC1783 - - -
Listeria innocua KC1784 - - -
Streptococcus pyogenes
SS482 - - -
Streptococcus pyogenes
SS91 - - -
Campylobacter jejuni D133 +/- +/- -
Campylobacter jejuni L1 +/- +/- -
Campylobacter jejuni L2 +/- +/- -
Campylobacter jejuni L9 +/- +/- -
Campylobacter fetus D223 - - -
Campylobacter fetus D373 - +/- -
Campylobacter fetus D406 - - -
Campylobacter fetus D411 - - -
Vibrio cholerae 01 Inata - - -
Salmonella typhimurium
2489-88 - - -
Clostridium difficile
0 880-221 - - -
Clostridium perfringens
860386 - - -
Clostridium septicum 70426 - - -
Streptococcus salivarius
SS908 - - -
Streptococcus salivarius
SS1062 - - -
__________________________________________________________________________
Top